蛋白質的功能和它的結構息息相關，而β-hairpins因其簡單的結構以及獨特的折疊特型而使它成為最廣泛的蛋白質摺疊研究標的。對於β-hairpin胜肽HP7而言，G8A突變會降低摺疊的速率，而N4A突變則會降低結構的穩定性，上述差異亦說明了蛋白質摺疊的複雜性。為了解突變對HP7摺疊的影響，我們運用分子模擬(MD)與多種進階取樣方法，包括：metadynamics, replica exchange molecular dynamics, 及forward flux sampling，來了解β-hairpins折疊的熱力學與動力學特性。當溫度升高時，HP7和G8A的Trp3-Trp10的T型堆積比例隨之遞減，而N4A則是會劇烈的下降。自由能分析顯示HP7和G8A單體在298 K以及343 K均可維持穩定的β-hairpins結構，代表兩者有很好的熱穩定性。此結果可以佐證圓二色光譜(CD)中216 nm的變化在低濃度條件下對溫度的敏感度比較低。而N4A在所有模擬溫度下都為不穩定的β-hairpin結構且較偏好無序的結構，亦和CD的結果相符。為了更進一步探討濃度效應，我們利用REMD分析β-hairpin在不同溫度下的二聚化行為。結果顯示HP7和G8A在兩個胜肽鏈之間形成的β-sheet比例隨溫度升高而減少，吻合實驗上CD圖譜在不同濃度間的差異。針對β-hairpin摺疊機制，透過MD的分析我們推敲其可能機制：β-turn先形成後，透過拉鍊機制形成主鏈氫鍵，形成完整的β-hairpin。基於此機制，運用FFS計算折疊速率，結果顯示β-turn的形成是整個折疊過程的關鍵中間產物。由於Ala側鏈的立體效應，G8A突變體會大幅降低β-turn的形成速率進而減緩折疊速率。但G8A突變對於後續氫鍵的形成並無顯著影響，因而導致了HP7和G8A的β-hairpin結構有相似的熱力學穩定性。

The functions of proteins are highly connected with the unique 3D structures. Due to its simple conformation and unique folding behaviors, β-hairpins are one of most widely used proteins for studying protein folding. For the β-hairpin peptide HP7, the G8A mutation is known to significantly reduce the folding rate of the β-hairpin structure. In contrast, the N4A mutation greatly destabilizes the β-hairpin conformation. These results demonstrate the complexity of the β-hairpin folding. To understand the effects of mutation on HP7 folding, we combined molecular dynamics (MD) simulations with advanced sampling techniques, including metadynamics, replica exchange molecular dynamics (REMD) and forward flux sampling (FFS), to characterize the thermodynamics and kinetics of the β-hairpin folding.
As temperature increases, the stacking probabilities of the Trp3-Trp10 pair within HP7 and the G8A mutant gradual decrease while that within the N4A mutant shows greater reduction. The free energy analyses illustrate that the β-hairpin conformations for both HP7 and G8A monomer remain stable at both 298 K and 343 K, suggesting very good thermostability for both peptides. These results agree with experimental circular dichroism (CD) analyses where the β-hairpin signal at 216 nm is less temperature sensitive at low concentrations. In contrast, the N4A mutant prefers disordered conformation at all tested temperatures, also consistent with experimental CD results. To further examine the concentration effect, we utilized REMD to analyze the peptide dimerization at various temperatures. Our results suggest that the content of inter-peptide β-sheets for HP7 and G8A gradually decreases with increasing temperature, leading to reduce β-sheet signal in CD thermogram observed in experiment. MD data further reveals that, for both HP7 and G8A, the turn structure forms first, followed by the formation of complete β-hairpin via the zipping mechanism. The FFS analyses based on such mechanism further identify the formation of turn as the key intermediate during the β-hairpin folding. G8A mutation drastically reduces the rate of β-turn formation due to the steric effect of the Alanine side chain. Note, however, the G8A mutation has little influence on the hydrogen bonds between β-sheets, leading to similar structural stabilities between HP7 and G8A.

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